Gabapentin Disrupts Binding of Perlecan to the α2δ1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation.

Our understanding of how osteocytes, the principal mechanosensors within bone, sense and perceive force remains unclear. Previous work identified "tethering elements" (TEs) spanning the pericellular space of osteocytes and transmitting mechanical information into biochemical signals. While we identified the heparan sulfate proteoglycan perlecan (PLN) as a component of these TEs, PLN must attach to the cell surface to induce biochemical responses. As voltage-sensitive calcium channels (VSCCs) are critical for bone mechanotransduction, we hypothesized that PLN binds the extracellular α2δ1 subunit of VSCCs to couple the bone matrix to the osteocyte membrane. Here, we showed co-localization of PLN and α2δ1 along osteocyte dendritic processes. Additionally, we quantified the molecular interactions between α2δ1 and PLN domains and demonstrated for the first time that α2δ1 strongly associates with PLN via its domain III. Furthermore, α2δ1 is the binding site for the commonly used pain drug, gabapentin (GBP), which is associated with adverse skeletal effects when used chronically. We found that GBP disrupts PLN::α2δ1 binding in vitro, and GBP treatment in vivo results in impaired bone mechanosensation. Our work identified a novel mechanosensory complex within osteocytes composed of PLN and α2δ1, necessary for bone force transmission and sensitive to the drug GBP.

Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser.

Two-color multiphoton microscopy through wavelength mixing of synchronized lasers has been shown to increase the spectral window of excitable fluorophores without the need for wavelength tuning. However, most currently available dual output laser sources rely on the costly and complicated optical parametric generation approach. In this report, we detail a relatively simple and low cost diamond Raman laser pumped by a ytterbium fiber amplifier emitting at 1055 nm, which generates a first Stokes emission centered at 1240 nm with a pulse width of 100 fs. The two excitation wavelengths of 1055 and 1240 nm, along with the effective two-color excitation wavelength of 1140 nm, provide an almost complete coverage of fluorophores excitable within the range of 1000-1300 nm. When compared with 1055 nm excitation, two-color excitation at 1140 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (for example, tdKatushka2). We demonstrate multicolor imaging of tdKa-tushka2 and Hoechst 33342 via simultaneous two-color two-photon, and two-color three-photon microscopy in engineered 3D multicellular spheroids. We further discuss potential benefits and applications for two-color three-photon excitation. In addition, we show that this laser system is capable of in vivo imaging in mouse cortex to nearly 1 mm in depth with two-color excitation.